Target-cell killing is one of the core endpoints of TCE functional evaluation. Compare: antigen-positive vs antigen-negative cells, TCE concentration-response, different E:T ratios, and time-dependent response; and obtain parameters including EC₅₀, maximal killing, killing kinetics, target dependence, and assay reproducibility.
| Method | Readout Principle | Applicable Window | Role |
|---|---|---|---|
| Luciferase target-cell luminescence | Luminescence decay of live cells, linear with live-cell number | 4–72 h, 96/384-well | Primary screen (default in this protocol) |
| Absolute-count flow cytometry (CTV + counting beads) | Reduction in absolute number of surviving target cells | 4–72 h | Confirmation and mechanistic analysis |
| LDH release | Release of intracellular enzyme from dead cells | 24–48 h | Low-cost supplement; must subtract effector-cell spontaneous-death background |
| Calcein-AM release | Release of preloaded fluorescent dye from target cells | 2–6 h | Supplement for rapid killing phase |
| Real-time live-cell imaging (IncuCyte-type) | Continuous loss of fluorescent target cells ± caspase-3/7 substrate | 0–72 h continuous | Killing kinetics curves (complementary to T₅₀/AUC) |
Cytotoxicity distinguishes between "burst-like single-round killing" and "sustained serial killing capacity": a high-quality molecule can kill efficiently even at low E:T ratios of 1:1 or even 1:2. In addition to the standard 10:1 primary endpoint, the protocol adds an E:T = 1:1 condition and multi-round serial killing (fresh target cells replenished every 2–3 days, 3–5 rounds). Killing collapse at low E:T (effective at 10:1 but failing at 1:1) and Emax plateau (<50%) both belong to the "insufficient killing depth" failure mode.
Target cells stably express luciferase and luminesce only when viable; killing reduces luminescence linearly with the number of live cells. In systematic comparative studies of cytotoxicity assays, this method has shown high sensitivity and good linearity[7]. Commercial luciferase-labeled target-cell lines are already available for TCE cytotoxicity screening and serve as the standard readout for TCE format screening[8][15]. After adding substrate per well, plate-based luminescence reading directly supports 96/384-well throughput and full curve fitting across concentration gradients. If target cells are difficult to stably transduce or primary target cells are used, fall back to flow cytometry.
A defining feature of BiTE-type molecules is rapid killing, which is already significant by 4–6 h. Set the time course at 4, 6, and 24 h (24 h as primary endpoint); killing speed directly relates to in vivo tumor clearance efficiency and CRS kinetics, and both metrics are included in the candidate profile. In addition to EC₅₀/Emax, add:
Product list for this module is being finalized and will be published in a subsequent update.
Epcoritamab in vitro cytotoxicity EC₅₀ against B-cell lymphoma cell lines ranges from 0.2 to 3.5 pM. Resting T cells obtained by GeneMedi negative selection show <10% killing in the absence of target antigen, with a target-dependence index >7.5, ensuring that the true target dependence of candidates is accurately assessed and preventing "false-positive" candidates from advancing into later development.
